Interest in biomolecules including proteins and oligonucleotides has exploded in recent years, as evidenced by the rise of the biotechnology industry. These materials are being intensively studied in a variety of ways and for many purposes, ranging from identification of individual components of complex biological mixtures through determination of Quantitative Structure Efficacy Relationships (QSER) to identification of biopharmaceutical drug candidates.
While supplies of raw materials are relatively abundant, an ongoing problem encountered in this work is separation and/or purification of these materials. Chromatographic methods are usually the method of choice, but the separations can be very challenging. Natural sources of biomolecules yield complex mixtures of materials with relatively low concentration of the desired product. Synthetic techniques produce crude products that may be contaminated by closely related impurities, for example, proteins differing in composition by only a few (or even a single) amino acid. Therefore, development of efficient and cost-effective methods for separation and purification of biomolecules is increasingly important.
Displacement chromatography can be used to perform such difficult separations in an efficient and cost effective manner. A particularly attractive feature of displacement chromatography is the ability to concentrate components of a mixture during the separation. Because of this combination of features, the technique is especially appropriate for industrial process-scale chromatography.
The key operational characteristic that distinguishes displacement from step elusion or step gradient chromatography is the use of a displacer compound that has greater affinity for these stationary phase than the desired product. The displacer competes for absorption sites on the stationary phase, causing the feed components to exit the column as adjacent “square wave” zones of highly concentrated pure material, in the order of increasing affinity of absorption. An important distinction between displacement and step gradient chromatography is that the displacer front always remains behind the adjacent feed zones in the displacement train, while, desorbents, for example, salts and organic modifiers, move through the feed zones. The implications of this distinction are quite significant in that displacement chromatography can concentrate and purify components from mixtures having low separation factors, while in the case of desorption chromatography, relatively large separation factors are generally required to give satisfactory resolution.
A limited number of materials for use as displacers in chromatographic systems have been described in the patent and scientific literature. These include large polyelectrolytes as displacers for separation of proteins in ion exchange systems. In addition, several types of low-molecular weight displacers for ion exchange have also been described. (See, for example, U.S. Pat. Nos. 5,478,924; 5,606,033 and 6,239,262). Low-molecular weight displacers have significant operational advantages as compared to large polyelectrolyte displacers. First and foremost, if there is any overlap between the displacer and the protein of interest, these low-molecular weight materials can be readily separated from the purified protein during post-displacement processing using standard size-based purification methods, for example, size exclusion chromatography and/or ultra filtration. This advantage is particularly important for meeting FDA standards for validating displacement chromatic graphic bioprocesses. The salt dependent adsorption behavior of these low-molecular weight displacers greatly facilitates column regeneration. Finally, the use of low-molecular weight displacers enables the operation of displacements in the selective displacement mode which can result in elution of the weakly retained proteins in the induced salt gradient, displacement of the bioproduct of interest and closely related impurities, and desorption of the more strongly retained impurities after breakthrough of the displacer front.
However, the major obstacle to the implementation of displacement chromatography has been a lack of displacer molecules for particular separations. Therefore, there is a continuing need for additional displacer compounds that have high affinity for the stationary phase of ion exchange chromatographic systems, as well as chemically selective displacers for specific separations.